In situ assay of substance in biological sample using labeled probe

ABSTRACT

A method for analyzing an objective substance, comprising reacting a labeled probe with an objective substance on a biological sample, said probe comprising a label substance of the formula (I):  
                 
 
     wherein A 1  is an aromatic group, R 1 is a hydrogen or —COCH 2 COC n F 2n+1  and n is an integer of 1-6, which is bonded to a probe selected from the group consisting of nucleic acid, nucleic acid binding protein, low molecular ligand and receptor for ligand (except antibody) to give a fluorescent complex, reacting the complex with an objective substance on a biological sample and assaying fluorescence of the resultant fluorescent complex, a labeled nucleic acid probe and a labeled nucleotide. According to the method of the present invention, defects such as hindrance of fluorescence due to contaminant substance, low sensitivity and the like can be resolved, thereby enabling analysis on a tissue.

FIELD OF THE INVENTION

[0001] The present invention relates to a novel in situ assay method foran objective substance in a biological sample, comprising assaying onsaid biological sample, a reagent therefor, particularly, a novellabeled nucleic acid probe and a fluorescent complex comprising saidprobe and a heavy metal ion, a labeled nucleotide for preparing saidlabeled nucleic acid probe and a process for preparing said labelednucleic acid probe. More particularly, the present invention relates toa novel method which can be preferably used for analyzing the functionand behavior of a certain substance (e.g., nucleic acid) on a biologicalsample (e.g., a biological tissue and a cell), by assaying thelocalization or concentration thereof on the biological sample, as wellas a labeled probe and a reagent for analysis which contains said probeto be used for said method.

BACKGROUND OF THE INVENTION

[0002] In the research field of life science and the field of clinicaldiagnostic and clinical tests, fluorescent substances have been widelyused as a label substance, besides radioactive substances, enzymes andthe like. With the progress of the image analyzing technique systems inrecent years, they have been more increasingly used in a broad range ofapplications, thereby providing new findings in the function andbehavior of biological substances in a living body.

[0003] Such fluorescent substances typically include compoundscomprising fluorescein, dansyl group, anthraniloyl group, pyrene,rhodamine, nitrobenzoxadiazol and the like.

[0004] The fluorescent substances, which are intercalated in betweendouble strands of nucleic acid (DNA) and enable fluorescent staining ofthe DNA, include Hoechst 33342 manufactured by Molecular Probe,4′,6′-diamino-2-phenylindol dihydrochloride (DAPI), propidium iodite(PI), acridium orange and the like. Besides these, commerciallyavailable products such as SYTO (TM), BOBO (TM), POPO (TM), TOTO (TM),YOYO (TM) and the like are used similarly.

[0005] To label lipids, fluorescent substances, such as4-nitrobenzene-2-oxa-1,3-diazol (NBD) and4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY), are used.

[0006] In recent years, fluorescent substances capable of labelingvarious ions or other low molecular substances (e.g., fura-2, indo-1,fluo-3, etc. for calcium ion, SBFI, etc. for sodium ion, mag-fura-2,mag-indo-1, etc. for magnesium ion, TSQ, etc. for zinc ion, SPQ, etc.for chloride ion and FICRhR for cyclic AMP) have been developed, and thebehavior of ions in a living body has been studied using thesefluorescent substances.

[0007] In an assay of a substance in a biological sample, it is desiredof a fluorescent substance, theoretically and practically, that (1) itdoes not deactivate nucleic acid, peptide, low molecular ligand and thelike after binding, (2) it has a high fluorescence quantum yield andhigh photostability, (3) its fluorescence lifetime is long, (4) it isfree of the effect of other endogenous fluorescent substances in thebiological sample, (5) it does not react non-specifically with anendogenous molecule in the biological sample, (6) it easily dissolves inwater and (7) its determination is convenient. Particularly, in an insitu assay on a tissue or cell, a fluorescent substance is furtherrequired to not react non-specifically with a biomolecule present in thetissue or cell or on the surface thereof.

[0008] However, some of the above-mentioned fluorescent substances areunstable to light and/or heat, some have low quantum yield, and othershave short excited fluorescent lifetime and are subject to the effect ofautofluorescence of other endogenous substances. The conventionallyknown fluorescent substances are not ideal fluorescent compounds, butrather, are insufficient, since they are more or less problematic in oneor more aspects, such as low S/N ratio, short fluorescence wavelengthand the like.

[0009] The influence of endogenous fluorescence in the assay ofsubstances in a liquid sample such as body fluid and cell extract can beremoved by using, as a lanthanoide metal-containing fluorescent complex,a complex labeled with a novel fluorescent substance and consisting of asubstance having affinity for the objective substance and an europiumion. A method has been developed which is free of an influence of thebackground fluorescence derived from a fluorescent substance ornon-fluorescent substance in a biological sample, particularly a serum,during assay of a physiologically active substance in the sample, andwhich comprises subjecting the complex to a time-resolved fluorescenceassay.

[0010] For example, use of a diazophenyl-EDTA-europium complex orisothiocyanatephenyl-EDTA-europium complex for an immnoassay has beenknown (Anal. Biochem, 137,335-343, 1984). In this immnoassay,β-naphthoyltrifluoroacetone (β-NTA) is added to the assay system in theco-existence of β-diketone and tri-n-octyl-phosphine oxide (TOPO) toachieve the highest sensitivity.

[0011] This assay system has been known as a DELFIA system (DissociationEnhanced Lanthanide Fluoroimmunoassay). A method utilizing a europiumcomplex represented by this system is advantageous in that an assaytarget in a biological sample can be detected without the influence offluorescence having a short lifetime which is derived from acontaminating substance in the body, due to the fluorescent property ofthis complex that its life is long.

[0012] On the other hand, the DELFIA system is associated with thedefect caused by a reaction between β-NTA or TOPO used for the assay andeuropium in a sample or in the environment, thereby producing strongfluorescence, which may prevent detection of the assay target.

[0013] In addition to the inherent defect this system has in that it issusceptible to the influence of the contaminated europium, the need toadd a fluorescence intensifier such as β-NTA makes the assay on a solidphase unattainable. There is also a problem of manipulative complexitydue to the step of adding a fluorescence intensifier. In conclusion, insitu assay of a physiologically active substance (e.g., nucleic acid,receptor, sugar chain, ganglioside and the like) fixed on a tissue orcell (surface) by this system is extremely difficult.

[0014] To resolve the above-mentioned defects of the DELFIA system, aCyber Fluor system that uses a complex of4,7-bis-(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid(BCPDA) and europium is known (Anal. Chem., 61, 48-53, 1989).

[0015] The use of BCPDA has made a great advancement in that manyeuropium fluorescent complexes can be introduced without a quenchingphenomenon (quenching phenomenon strikingly decreases the fluorescencequantum yield) caused when one probe is labeled with many fluoresceinsand that it is highly stable and can resolve the defects of the DELFIAsystem.

[0016] However, the Cyber Fluor system has a fatal defect in that itssensitivity is lower than that of the DELFIA system by the order of twodigits or more. To compensate for the defect, synthesis of a number ofeuropium complexes was tried, and, for example, trisbipyridine cryptate(TBP) europium complex and the like are known (Clin. Chem., 196-201,1993, U.S. Pat. No. 5,262,526, JP 07-10819 A and the like). These newlydeveloped europium fluorescent complexes have defects in that they haveshort excitation wavelengths and weak fluorescence, and they requiremany synthetic steps. Thus, they do not have particularly superiorproperty as compared to the above-mentioned two europium fluorescentcomplexes.

[0017] Many studies have been made so far with respect to europiumfluorescent complex and it has been found that β-diketone-europiumfluorescent complex has greater fluorescence intensity than aromaticamine-europium complex, and of the β-diketone ligands, a europiumfluorescent complex of 2-naphthoyltrifluoroacetone (β-NTA) and2-thenoyltrifluoroacetone (TTA) particurlaly has the greatestfluorescence intensity.

[0018] The present inventors synthesized various β-diketonato-europiumTOPO complexes to study the effect of β-diketone as a substituent on thefluorescence property of the β-diketone-europium fluorescent complex,and found that the fluorescence intensity of these complexes isdependent on the composition and structure of the substituents R¹ and R²of the β-diketonato (R¹COCH₂COR²). In other words, when R¹ is anaromatic hydrocarbon residue, stronger electron attractiveness of R²results in stronger fluorescence intensity of the complex, based onwhich finding an immunoassay utilizing a β-diketone type europiumfluorescent complex having a dramatically improved fluorescenceintensity has been found (U.S. Pat. No. 5,859,297 and Anal. Chem., 70,596-601, 1988).

[0019] However, the use of this β-diketone type europium fluorescentcomplex for the assay of a substance having various actions that is on abioloical tissue or cell, such as a physiologically active substance,has not been disclosed. Many difficulties are foreseeable in an assay ona tissue or cell of a physiologically active substance in the biologicalsample, for example, a great influence of contaminating substance, adifficult high sensitivity assay, an unattainable easy assay and thelike.

[0020] It is therefore an object of the present invention to provide ameans of resolving defects such as hindrance of fluorescence by acontaminating substance and low sensitivity, so that a substance in atissue or cell or on surface thereof, such as nucleic acid, nucleic acidbinding protein, receptor, sugar chain, ganglioside and the like can beassayed as it is on the tissue or cell with high precision and highsensitivity.

SUMMARY OF THE INVENTION

[0021] The present invention is based on the finding that a β-diketoneform europium fluorescent complex has superior characteristics as alabel for probe for the high sensitivity assay of a physiologicallyactive substance such as nucleic acid, nucleic acid binding protein,receptor, enzyme, sugar chain, ganglioside and the like on a tissue orcell, since it has a noticeably long fluorescence lifetime and permitstime-resolved fluorescence assay, assay upon elimination of blankfluorescence, use in one step and has a, long wavelength fluorescencelifetime.

[0022] Accordingly, the present invention provides a method foranalyzing a biological substance comprising the use of a label substanceof the following formula (I):

[0023] wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II)

[0024] wherein A² and A³ are the same or different and each is anaromatic group, R² and R³ are the same or different and each is ahydrogen or COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, reagentstherefor and a preparation method thereof. More particularly, thepresent invention provides the following.

[0025] (1) A method for analyzing an objective substance, comprisingreacting a labeled probe with an objective substance on a biologicalsample, said probe comprising a label substance of the formula (I) or alabel substance of the formula (II) bonded to a probe selected from thegroup consisting of nucleic acid, nucleic acid binding protein, lowmolecular ligand and receptor for ligand (except antibody) via across-linking group or a cross-linking group and a conjugating group,adding a heavy metal ion and assaying fluorescence of the resultantfluorescent complex.

[0026] (2) The method for analyzing an objective substance, comprisingadding a heavy metal ion to a labeled probe, said probe comprising alabel substance of the formula (I) or a label substance of the formula(II) bonded to a probe selected from the group consisting of nucleicacid, nucleic acid binding protein, low molecular ligand and receptorfor ligand (except antibody) via a cross-linking group or across-linking group and a conjugating group to give a fluorescentcomplex, reacting the complex with an objective substance on abiological sample and assaying fluorescence of the resultant fluorescentcomplex.

[0027] (3) A labeled nucleic acid probe comprising a label substance ofthe formula (I) or a label substance of the formula (II) bonded to anucleic acid probe via a cross-linking group.

[0028] (4) A fluorescent complex comprising the labeled nucleic acidprobe of (3) and a heavy metal ion.

[0029] (5) A reagent for analyzing a nucleic acid, comprising thelabeled nucleic acid probe of (3).

[0030] (6) A labeled nucleotide comprising a label substance of theformula (I) bonded to a nucleotide via a cross-linking group.

[0031] (7) A fluorescent complex comprising the labeled nucleotide of(6) and a heavy metal ion.

[0032] (8) A method for producing a labeled nucleic acid probecomprising reacting the labeled nucleotide of (6), dNTPs and a singlestrand DNA in the presence of a DNA polymerase.

[0033] (9) A method for producing a labeled nucleic acid probecomprising reacting the labeled nucleotide of (6), dNTPs and a doublestranded DNA in the presence of 5′-exonuclease, DNase and a DNApolymerase.

[0034] (10) A labeled nucleic acid probe obtained by the productionmethod of (8) or (9).

[0035] (11) A reagent for analyzing nucleic acid comprising a labelsubstance of the formula (I) or the formula (II), to which avidin iscovalently bonded via a cross-linking group (hereinafter to be referredto as label substance A) and a nucleotide to which biotin is bonded viaa linkage group (hereinafter to be referred to as nucleotide B).

[0036] (12) A method for analyzing an objective substance comprisingreacting the objective substance with a nucleic acid probe comprisingthe nucleotide B as a component on a biological sample and then with thelabel substance A, adding a heavy metal ion and assaying thefluorescence of the resultant fluorescent complex.

[0037] According to the method of the present invention, defects such ashindrance of fluorescence by a contaminating substance and lowsensitivity can be resolved in the analysis of nucleic acid, nucleicacid binding protein, receptor, sugar chain, ganglioside and the like ona biological tissue, cell or chromosome in a biological sample. Inparticular, hindrance due to contamination with a lanthanoide metal ionin a sample or environment can be removed, and assay of the objectivesubstance with high sensitivity and with small steps.

DETAILED DESCRIPTION OF THE INVENTION

[0038] The label substance in the present invention is represented bythe formula (I) or the formula (II). In the formulas, A¹, A² and A³ arethe same or different and each is a trivalent aromatic group,particularly a conjugated double bond, wherein when R¹, R² or R³ ishydrogen, A¹, A² or A³ it binds with is a divalent aromatic group. Suchdivalent or trivalent aromatic group is exemplified by

[0039] and the like. Those having a substituent to these aromatic rings,such as methylphenylene and methyldibenzothiopehne, are alsoexemplified.

[0040] Particularly preferable aromatic group is the following:

[0041] R¹, R² and R³ are each independently hydrogen orCOCH₂COC_(n)F_(2n+).

[0042] In the formulas (I), (II) and (III), n and n at R¹, R² and R³ arean integer of 1-6, preferably 2-4.

[0043] In the present invention, a particularly preferable labelsubstance is represent ed by the formula (III):

[0044] wherein n is an integer of 1-6.

[0045] In the present invention, the probe is selected from the groupconsisting of nucleic acid, nucleic acid binding protein, ligand andreceptor for ligand (except antibody).

[0046] In the present invention, the objective substance is a componentin the biological sample, which is to be the subject of the analysis.Preferable examples thereof include nucleic acid, nucleic acid bindingprotein, ligand, receptor for ligand and the like.

[0047] The nucleic acid binding protein is a protein that specificallybinds with the nucleic acid having a specific nucleotide sequence, suchas histone, DNA binding protein, Lac I protein and the like. By the useof a transcription regulating factor of cytokine (a kind of DNA bindingproteins), such as NF-κB and the like, as a probe to be labeled, theinteraction between the transcription factor and DNA can be visualized.

[0048] The low molecular ligand here means an organic compound such assugar chain, aromatic compound, ganglioside, oligosaccharide, peptideconsisting of 2-10 amino acids and the like. Examples thereof includemyc peptide, thyroxine, triiodothyronine, ganglioside GM2, cellobiose,sugar chain having a sialic acid at the end thereof and the like.

[0049] The receptor for ligand means a substance that specifically bindswith a specific ligand that is located on or in a cell or between cells,such as cellulose binding protein, sialic acid binding lectin, albuminreceptor and the like.

[0050] Further examples of the low molecular ligand or receptor includehormone or hormone receptor such as insulin, insulin receptor, EGF, EGFreceptor, HGF, HGF receptor, TSH, TSH receptor and the like, andreceptors of low molecular ligands such as receptor of cytokine (e.g.,IL-8 and the like) or chemokine, acetylcholine receptor, histaminereceptor and the like.

[0051] The protein kinase C can bind with the derivative of phorbolester and can be assayed by the method of the present invention. Inaddition, the enzymes such as cAMP-dependent protein kinase,cGMP-dependent protein kinase, calmodulin-dependent phosphoenzyme,tyrosine-phosphorylated enzyme and the like can be assayed by way ofligand-receptor reaction, wherein the labeled probe of the presentinvention can be used as the probe for the substrate binding site of theenzyme.

[0052] Various lectins against various sugar chain and ganglioside canbe used as the probe of the present invention. Examples of lectininclude concanavalin A against D-mannose bonded with various proteins onthe cell, wheat germ agglutinin against di-N-acetylchitobiose, sialicacid binding lectin against sialic acid, which is derived from Limuluspolyphemus and the like.

[0053] Examples of nucleic acid and nucleic acid probe include DNAshaving a series of various deoxyribonucleic acids (dATP, dGTP, dTTP,dCTP, dUTP) and RNAs having a series of various ribonucleic acids (rATP,rGTP, rTTP, rCTP, rUTP). These nucleic acid probe has a nucleotidesequence consists of cDNA or antisense oligonucleotides thatspecifically hybridizes with mRNA which expresses in the cell.Alternatively, a nucleic acid probe having a nucleotide sequencecomplementary to a part of a specific sequence of nucleic acid orchromosome in the cell can be used.

[0054] Examples of the nucleic acid or gene on chromosome in the cellinclude oncogene (e.g., abl, erb, fos, myb, myc, ras, src and the like),tumor suppressor gene (e.g., p53 and the like), rearranged T cellreceptor gene, rearranged immunoglobulin gene, a part of the nucleotidesequence of pathogenic virus gene such as Epstein-Bar virus (EBV),herpes simplex virus (HSV), cytomegalovirus (CMV), hepatitis B virus(HBV), rotavirus, adenovirus and the like, a part of the nucleotidesequence of infectious pathogenic microorganism gene such as malariaprotozoa, fungus, mycoplasma and the like, and nucleic acid having anucleotide sequence complementary thereto.

[0055] A part or the whole of the nucleic acid probe complementary tothese genes or homologous therewith may have a modified group such asmethyl group and the like as long as it does not affect bonding with alabel substance.

[0056] The labeled nucleic acid probe of the present invention is acompound having affinity for a specific substance particularly on thetissue or cell, such as nucleic acid, nucleic acid binding protein andthe like containing the above-mentioned genes on chromosome and thelike.

[0057] The labeled probe in the present invention consists of a probeselected from the group consisting of nucleic acid, nucleic acid bindingprotein, low molecular ligand and receptor for ligand (except antibody)and a label substance bonded thereto. The labeled nucleic acid probe ofthe present invention consists of a nucleic acid and a label substancebonded to each other. The labeled nucleotide of the present inventionconsists of a nucleotide and a label substance bonded thereto. The bondbetween the label substance and the probe or nucleotide is a bond via across-linking group. It may be a covalent bond via a conjugating group.

[0058] The cross-linking group is via a bond between a label substanceand a conjugating group, probe, nucleotide or avidin. That is, in alabeled probe and a labeled nucleotide having a conjugating group, theconjugating group exists between the cross-linking group and the probeor nucleotide.

[0059] The label substance A in the present invention consists of avidinand a label substance bonded via a cross-linking group. The bindingratio of avidin-label substance is 1-50, preferably 2-30. The nucleotideB in the present invention consists of biotin and nucleotide bonded viaa linkage group.

[0060] Avidin in the present invention is a glycoprotein that iscontained in the egg white and specifically binds with biotin. Avidinmay be a streptoavidin derived from a microorganism (genusStreptococcus) or a recombinant protein thereof.

[0061] Biotin in the present invention is a substance called vitamin Hand coenzyme R and binds extremely firmly with avidin or streptoavidin,wherein the bonding strength is far greater than the bond of typicalimmunoconjugate.

[0062] The cross-linking group in the present invention is derived froma group capable of bonding with both nucleic acid, nucleic acid bindingprotein, low molecular ligand, receptor for ligand, nucleotide or avidinand aromatic group. Alternatively, it is derived from a group capable ofbonding with both linkage group and aromatic group.

[0063] Examples of the cross-linking group include —NH—CS—, —NH—CO—,—CO—, —N₂—, —NH—, —SO₂—, —CH₂—S—, —CH₂—NH—, —(CH₂)₆—NH—CO—CH₂—CH₂—CO—,and S—S- and the like. Particularly preferable cross-lining groups aresulfonyl group and carbonyl group.

[0064] The linkage group in the present invention is free of particularlimitation as long as it connects a cross-linking group and a nucleicacid, nucleic acid binding protein, low molecular ligand, receptor forligand or nucleotide. Preferable linkage group is a divalent aliphatichydrocarbon group having 5-25 carbon atoms and 7 or less amide bondsbetween carbons. Specific examples include a group of the formula (IV):

[0065] wherein a is an integer of 0-6 and b is 0 or 1.

[0066] Another preferable mode of the linkage group is a linkage groupcontaining biotin and avidin through affinity binding.

[0067] Biotin affinity binding with avidin is preferably further bondedto a probe via a linkage group. Examples of preferable linkage groupinclude divalent aliphatic hydrocarbon group having 5 to 25 carbon atomsand optionally having 7 or less amide bonds between carbons.Specifically, it is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂—, whereinpreferable bond is(probe)—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂-biotin:avidin-(cross-linkinggroup-label substance).

[0068] The linkage group binding biotin and nucleotide in nucleotide Bis free of limitation as long as it binds biotin and nucleotide.Preferable linkage group include a divalent aliphatic hydrocarbon grouphaving 5 to 25 carbon atoms and optionally having 7 or less amide bondsbetween carbons. Specifically, it is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂—, whereinpreferable bond is(nucleotide)—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂-(biotin).

[0069] When the label substance of the formula (II) is bonded to probeor avidin via two cross-linking groups, the two cross-linking groups maybe the same or different. Again, when it is bonded to probe or avidinvia two conjugating groups, the two conjugating group may be the same ordifferent.

[0070] The label substance of the formula (II) can be used upon bindingto two probes or avidin. The two probes may be the same or different twoprobes or avidin. By binding to two probes, a synergistic binding effectcan be expected.

[0071] For example, one example of the labeled nucleic acid probe of thepresent invention is a label substance bonded to different nucleic acidprobes. These probes are nucleic acid probes having nucleotide sequencescomplementary to or homologous with the same or different genes. Inother words, a probe having plural nucleic acids recognizing thespecific sites of the assay target binds with the nucleic acid in thecell having nucleotide sequences complementary hereto or nucleotidesequences homologous thereto and becomes a so-called divalent probe byforming a fluorescent complex upon addition of a heavy metal ion (e.g.,lanthanoide metal ion), thereby affording a possible synergistic effect.

[0072] Even when the two binding probes are the same, each can bind withan objective substance having same plural specific sites. Thus, theeffect is not a simple addition but expected to be synergistic.

[0073] In the analysis method of the present invention, different kindsof labeled probes may be used simultaneously upon mixing.

[0074] While the binding ratio of the probe-label substance is free ofparticular limitation, it is generally 1-100, preferably 1-20.

[0075] The binding ratio of the nucleotide-label substance is free ofparticular limitation, it is generally 1-50, preferably 1-20.

[0076] The binding ratio of the avidin-label substance is free ofparticular limitation, it is generally 1-50, preferably 2-30.

[0077] The labeled probe, labeled nucleotide or label substance A can beproduced by the use of the following functional groups as long as itdoes not exert an adverse influence, for binding a label substance tothe probe, nucleotide or avidin. For example, various binding groupssuch as isothiocyanate group reactive with amino group, sulfonyl halidegroup (sulfonyl chloride group, sulfonyl fluoride group and the like),o-phthalaldehyde group in the presence of 2-mercaptoethanol,N-substituted maleimide group and the like for carbodiimide group andthiol group, iodoacetamide group for histidine and the like.

[0078] Specifically, a ligand, nucleic acid and the like and a labelingcompound of the following formula in a molar amount of 1-20 per mol ofligand, nucleic acid and the like are reacted in a solvent.

[0079] The present invention also relates to a fluorescent complexcontaining a labeled nucleic acid probe and a heavy metal ion. Examplesof the heavy metal ion include lanthanoide metal ion and radium ion,with preference given to lanthanoide metal ion. The lanthanoide metalion to be used in the present invention includes ions of europium (Eu),samarium (Sm), terbium (Tb), dysprosium (Dy) and the like. It istypically used in the form of a chloride, but may be used in the form ofother salts as long as the assay is not influenced. In the presentinvention, these lanthanoide metal ions may be used alone or incombination.

[0080] The reaction between the labeled probe and the objectivesubstance in the present invention is the reactions between nucleic acidand nucleic acid, nucleic acid and nucleic acid binding protein, andligand and receptor for ligand in a biological sample. For facilitatedreaction, the biological sample may be pre-treated. For example, nucleicacid extraction by AGPC, protein dissociation treatment with ethanol andthe like can be applied.

[0081] The biological sample is preferably a cell, tissue or chromosome.

[0082] The analysis method of the present invention analyzes theobjective substance in the cell and on the cell surface, wherein alabeled probe is reacted with the objective substance at a tissuesection, on a cell surface, on a chromosome and the like, a heavy metalion such as lanthanoide metal ion, radium ion and the like is added andfluorescence of the resultant complex is assayed, or a heavy metal ionsuch as lanthanoide metal ion, radium ion and the like is added to alabeled probe to give a fluorescent complex, which is reacted with theobjective substance on a biological sample and fluorescence of thecomplex after reaction is assayed.

[0083] The analysis method of the present invention may comprisereacting a nucleic acid probe containing nucleotide B as a componentwith the objective substance on a biological sample, then reacting withlabel substance A, adding a heavy metal ion, and assaying fluorescent ofthe resultant fluorescent complex.

[0084] The nucleic acid probe containing nucleotide B as a componentmeans that one or more nucleotides in the nucleotide sequence is(are)nucleotide B. Namely, it is a nucleic acid probe binding with biotin.

[0085] The nucleic acid probe containing nucleotide B as a component canbe obtained by reacting nucleotide B, dNTPs and single strand DNA in thepresence of a primer and a DNA polymerase to give a double stranded DNAand denaturing the obtained DNA with heat to give a single strand DNA.

[0086] The nucleic acid probe containing nucleotide B as a component canbe obtained by reacting nucleotide B, dNTPs and a double stranded DNA inthe presence of 5′-exonuclease, DNase and a DNA polymerase to give adouble stranded DNA and denaturing the obtained DNA with heat to give asingle strand DNA.

[0087] More specific analysis method is exemplified by the methodcomprising immersing a biological sample in a buffer containing alabeled probe, incubating the sample to allow reaction of the objectivesubstance and the labeled probe, washing off excess labeled probe withthe buffer, immersing the probe in a buffer containing lanthanoide metalion to form a complex and assaying the fluorescence of the resultantcomplex.

[0088] In addition, a method is exemplified, which comprises admixingbuffer containing lanthanoide metal ion with a buffer containing alabeled probe to form a complex, immersing a biological sample in thismixture, incubating the sample to allow reaction with the objectivesubstance, washing off excess (labeled probe:lanthanoide metal ion)complex and assaying the fluorescence of the resultant complex on thebiological sample.

[0089] As a different specific method, the following method isexemplified. That is, a double stranded DNA having a sequence to be theassay target, nucleotide B and dNTPs are reacted in the presence of5′-exonuclease, DNase and a DNA polymerase to give a biotin-boundnucleic acid probe. A biological sample is immersed in a buffercontaining this biotin-bound nucleic acid probe and incubated to allowreaction of the objective substance and the biotin-bound nucleic acidprobe, and excess biotin-bound nucleic acid probe is washed off. Then,the sample is immersed in a buffer containing the label substance A tobind biotin and avidin, and excess label substance A is removed. Then,the sample is immersed in a buffer containing lanthanoide metal ion toform a complex and the fluorescence of the resultant complex is assayed.

[0090] By these methods, the presence of the objective substance in abiological sample such as a tissue, cell, chromosome and the like isvisualized and analyzed for localization and concentration. In addition,abnormalities with respect to the objective substance can be analyzed.

[0091] The visualized image obtained by the use of the inventive labeledprobe can be retained through a fluorescence microscope, confocallaser-scanning microscope and the like. The fluorescence signal itselfis assayable with a fluorescence assay device, time-resolvedfluorescence assay device and the like.

[0092] In particular, the inventive labeled nucleic acid probe isreacted with a biological sample of a tissue, cell, chromosome and thelike and visualize the objective substance therein by colonyhybridization, fluorescence in situ hybridization (FISH) of tissue andchromosome, nucleic acid sandwich hybridization, comparative genomehybridization (CGH) and the like.

[0093] The present invention also relates to a labeled nucleotide. Thenucleotide of the present invention itself has affinity with a specificsubstance on a tissue or cell. It may be used to produce a labelednucleic acid probe by binding with a different nucleotide or by nicktranslation method from a double stranded DNA.

[0094] The nucleotide in the labeled nucleotide and nucleotide ofnucleotide B of the present invention is not particularly limited and isexemplified by ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, dTTP, dUTP and thelike, with particular preference given to dUTP.

[0095] The particularly preferable labeled nucleotide has the followingformula (V):

[0096] wherein X is a conjugating group of the formula (IV) and Y is asulfonyl group or carbonyl group, R is a group of the formula

[0097] and p is 0 or 1.

[0098] The present invention further relates to a fluorescent complexcontaining the above-mentioned labeled nucleotide and a heavy metal ion.Examples of the heavy metal ion include the above-mentioned lanthanoidemetal ion and radium ion, with preference given to the above-mentionedlanthanoide metal ion.

[0099] A labeled probe can be obtained by incorporating a labelednucleotide such as the labeled dUTP of the present invention and thelike, when synthesizing a fragmented probe DNA using DNA extracted fromthe tissue or cell, particularly chromosomal DNA. To be specific,labeled nucleotide, dNTPs and double stranded DNA is reacted in thepresence of 5′-exonuclease, DNase and a DNA polymerase to give a labelednucleic acid probe. Alternatively, a labeled nucleotide, dNTPs and asingle strand DNA are reacted in the presence of a DNA polymerase togive a labeled nucleic acid probe. Particularly preferably, labelednucleotide of the present invention, such as labeled dUTP and the likeis incorporated by nick translation method to give a DNA or a DNAfragment usable as a labeled nucleic acid probe.

[0100] Moreover, labeled nucleic acid probe, labeled nucleotide ornucleotide B of the present invention can be incorporated into DNA orRNA by nucleic acid amplification by PCR (polymerase chain reaction)method, LCR (ligase chain reaction) method, NASBA method and the like.The obtained DNA or RNA can be used for the analysis of the objectivesubstance as a labeled nucleic acid probe or a biotin-bound nucleic acidprobe.

[0101] The nucleic acid probe obtained by incorporating the labelednucleotide or nucleotide B of the present invention, that comprises aDNA complementary to the DNA of a tissue or cell can be particularlysuitably used for the analysis of abnormalities in the chromosome of theobjective tissue or cell.

[0102] The reagent for the analysis of nucleic acid of the presentinvention contains the above-mentioned novel labeled nucleic acid probeor labeled nucleotide. Preferably it contains a heavy metal ion such asthe above-mentioned lanthanoide metal ion, radium ion and the like.

[0103] The reagent for the analysis of nucleic acid of the presentinvention contains label substance A and nucleotide B. The reagentcontaining label substance A and nucleotide B preferably furthercontains dNTPs, primer, DNA polymerase and heavy metal. As a differentmode, a reagent containing label substance A and nucleotide B preferablyfurther contains dNTPs, 5′-exonuclease, DNase, DNA polymerase and heavymetal.

[0104] The present invention is explained in detail by illustrativereference examples and examples, to which the present invention is notlimited in any way.

REFERENCE EXAMPLE 1 Synthesis of 4,4′-diacetyl-o-terphenyl

[0105] To a solution of CH₂Cl₂ (200 ml), AlCl₃ (210 mmol) and CH₃COCl(205 mmol) was gradually added a solution of CH₂Cl₂ (100 ml) ando-terphenyl (100 mmol) dropwise with stirring at 0° C. The mixture wasstirred at 0° C. for 30 min and further stirred at room temperature for24 hr. The reaction solution was refluxed for 2 hr, and then poured intoconc. hydrochloric acid with ice. The mixture was sufficiently stirred,and CH₂Cl₂ was distilled away under reduced pressure. The precipitatewas separated by filtration, and thoroughly washed with water. Theproduct was recrystallized from 2-butanone (about 250 ml) to give needlecrystals, which were separated by filtration and dried in vacuo. Theyield was 22.1 g (70.3%). The results of elemental analysis were asfollows.

[0106] Element analysis: calculated: C %=84.05, H %=5.77 found: C%−84.96, H %=5.87

REFERENCE EXAMPLE 2 Synthesis of intermediate of labeled compound

[0107] The intermediate having the following structure was synthesized.

[0108] To anhydrous ether (Et₂O, 30 g) were added NaOCH₃ (3.0 g),4,4′-diacetyl-o-terphenyl (10 mmol) and C₃F₇COOC₂H₅ (20 mmol), themixture was stirred in a sealed container at room temperature for 24 hr.Anhydrous ether was distilled away to give a residue, which was dried invacuo for 30 min. The product was neutralized with 15% sulfuric acid(100 ml), and the precipitate was separated by filtration and washedwell with water. The precipitate was dissolved in ethanol (200 ml) underheating, and the insoluble substance was removed by filtration. Thesolution was concentrated to about 20 ml under reduced pressure. Thissolution was gradually added dropwise to petroleum ether (200 ml) understirring. The mixture was sufficiently stirred and filtered to remove asmall amount of deposited precipitate. The filtrate was evaporated tocompletely remove the organic solvents. The obtained oily substance wasdried in vacuo to give a yellow powder. The product was dried in vacuofor 24 hr. The yield was 460 g (65.0%).

[0109] Elemental analysis: calculated; C %=51.00, H %=2.28 found: C%=51.22, H %=2.61

[0110]¹H-NMR confirmed that the product was the objective compound.

REFERENCE EXAMPLE 3 Synthesis of labeled compound

[0111] The labeled compound having the following structure wassynthesized.

[0112] To chlorosulfuric acid (3.5 ml) was gradually added β-diketone(the intermediate obtained in Reference example 2, 2 mmol) understirring at room temperature. The reaction mixture was stirred at roomtemperature for 7 hr, then carefully added dropwise to ice water (150ml, outside cooled with ice water) under stirring. The resultantprecipitate was immediately centrifuged, washed with cold water (about5° C.) and centrifuged twice. The precipitate was suspended in a smallamount of cold water and transferred onto a glass filter, and water wasremoved by suction filtration. The resultant chlorosulfonylatedβ-diketone was dried in vacuo at room temperature for 48 hr or more. Theyield was 77%.

[0113] Elemental analysis: calculated: C %=44.76, H %=1.88 found: C%=44.50, H %=1.92

[0114]¹H-NMR confirmed that the product was the objective compound.

EXAMPLE 1 Labeling of p53, Human, Probe (exon 4 translated) a labeledcompound

[0115] The labeled compound obtained in Reference Example 3 and p53,Human, Probe (exon 4 translated) manufactured by Oncogene ResearchProduct (Cosmo Bio) were reacted in the following manner to prepare alabeled DNA wherein said labeled compound is bound with p53, Human,Probe mediated by a sulfonyl group.

[0116] One hundred pmol of p53, Human, Probe (exon 4 translated) wasdissolved in 0.1 mol/l carbonate buffer solution (pH=9.3, 100 μl). Tothis DNA solution was gradually added a solution (10 μl) of the labeledcompound having a mole number equal to said nucleic acid (having about280 amino groups/molecule) dropwise under stirring at room temperature.The mixture was stirred at room temperature for 1 hr, extracted withphenol/chloroform, and subjected to ethanol precipitation. Theprecipitate was washed with 80% ethanol and dried. The dried precipitatewas re-dissolved in 0.05 mol/l carbonate buffer solution (pH=8.0, 100μl).

[0117] The molarity of the labeled compound contained in this solutionwas calculated. In addition, the molar absorption coefficient of thelabeled compound at 330 nm was calculated from the absorbance at 330 nm.As a result, the absorption coefficient was 0.97 mol⁻¹cm⁻¹+. There wasno absorption by DNA at 330 nm. Assuming that molar absorptioncoefficient does not change during the process of labeling reaction, theconcentration of the label in the labeled DNA solution and the bindingratio of the label and DNA were calculated.

[0118] The binding ratio of DNA to the labeled compound obtained by theabove method was about 1.

EXAMPLE 2 Hybridization of the labeled DNA with genes in a liver tissue

[0119] A liver tissue excised from human was fixed with 4%paraformaldehyde-PBS at 4° C. overnight, and dehydrated with 70%, 80%,90% and 100% ethanol, successively. All water used in this experimentwas purified water treated with diethylpyrocarbonate (DEPC). Furtherdehydration was performed by exchanging the solution in the liver tissuetwice with 100% ethanol. Then, the liver tissue was transferred intoxylene, heated at 60° C. for 2 hr (3 times), and embedded with paraffin.

[0120] The section (about 5 μm) of this paraffin-embedded tissue wasprepared using a microtome, placed on a thoroughly washed slide glass,and dried at 37° C. for 6 hr to give a section preparation. The sectionpreparation was further dried with a drier, treated with xylene, 100%ethanol, 90% ethanol, 80% ethanol, 70% ethanol and phosphate buffer (PB,pH 7.4), successively, and treated with proteinase K solution (10 mMTris-HCl (pH 8.0), 1 mM EDTA; 10 μg/ml) for 20 min.

[0121] The section preparation was then immersed in 4%paraformaldehyde-PB solution for 10 min, washed with PB, and treatedwith 0.2N hydrochloric acid for 10 min, with PB for 1 minute, with 0.1 Mtriethanolamine hydrochloric acid (pH 8.0) for 1 minute, with 0.1 Mtriethanolamine hydrochloric acid (pH 8.0) containing 0.25% aceticanhydride for 10 min, and with PB for 1 minute. The section preparationwas further treated with 70% ethanol, 80% ethanol, 90% ethanol and 100%ethanol, successively, and air-dried to give an air-dried sample. Theair-dried sample was immediately subjected to the followinghybridization.

[0122] As a hybridization solution, a solution comprising 50% formamide,10 mM Tris-HCl (pH 7.6), 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1mM EDTA, 200 μg/ml tRNA and 1×Denhardt's solution was prepared.

[0123] The labeled DNA obtained in the Example was dissolved in thishybridization solution to the concentration of 5 ng/ml. The mixture(about 40 ml) was dropped onto the above air-dried sample. Prior tohybridization, the sample was prehybridized with the hybridizationbuffer without the labeled DNA at 37° C. for 2 hr. Then, the air-driedsample was covered with parafilm (CAN Co.), and incubated at 37° C. for16 hr in a moisture chamber, in which a paper towel moistened with 50%formamide solution was set, for hybridization with the labeled DNA.

[0124] After hybridization, the parafilm was removed from the slideglass in 5×SSC solution (40° C.), and the slide glass having the sectionhybridized with the labeled DNA was heated in 2×SSC, 50% formamide at40° C. for 30 min. The slide glass was washed with TNE solution (aqueoussolution containing Tris-HCl buffer, NaCl, and EDTA), and reacted with 5μg/ml RNase A in TNE solution for 10 min.

[0125] The slide glass was then washed with 2×SSC at 40° C. for 20 min(once), and 0.2×SSC at 40° C. for 20 min (twice). Finally, the slideglass was immersed in 0.2×SSC solution (pH 8.5) containing 0.1 mMeuropium chloride to give a fluorescent complex from the reaction of thelabeled DNA with europium chloride. The section on the slide glass wasobserved with a fluorescence microscope.

[0126] As a result, signals derived from the fluorescent complex of thelabeled DNA and europium chloride were detected on the section. Whenhybridization was performed using DNA labeled with fluoresceinisothiocyanate (FITC) in the place of the labeled DNA above as acomparative example, hybridization signals were hardly detected. In thisexperiment, europium chloride was not added to detect the signals.

EXAMPLE 3 Preparation of a labeled PCR product

[0127] Two oligonucleotides (SEO ID: NO: 1 and SEQ ID: NO: 2), havingthe nucleotide sequences homologous to the sense and antisense sequenceof hepatitis B virus surface antigen (HBsAg) gene, respectively, weresynthesized by phosphoamidite method using an automatic DNA/RNAsynthesizer. In the final step of the synthesis, a labeled compound wasreacted with the 5′ termini of the elongated oligonucleotides to givetwo kinds of labeled oligonucleotides shown in the following formulas(1) and (2).

[0128] wherein Q¹ is oligonucleotide (SEQ ID: NO: 1) in which the aminogroup of the nucleotide at the 5′ terminus binds to the sulfonyl groupof the labeled compound.

[0129] wherein Q² is oligonucleotide (SEQ ID: NO: 2) in which the aminogroup of the nucleotide at the 5′ terminus binds to the sulfonyl groupof the labeled compound.

[0130] Using the above two labeled oligonucleotides as a pair of primersand the nucleic acid fraction extracted with phenol/chloroform from theserum derived from a patient, who was strongly positive against HBsAg,as a template, PCR was carried out under the following conditions.

[0131] A reaction mixture consisting of 10 mM Tris-HCl (pH 9.0), 50 mMKCl, 1.5 mM magnesium chloride, 0.1% Triton X-100, dNTPs (50 μM each),0.02 U/μl Taq DNA polymerase, and primers (0.4 pM each) was used foramplification.

[0132] After preheating at 95° C. for 2 min, thermal denaturation wasperformed at 95° C. for 30 sec, annealing was performed at 57° C. for 30sec, and elongation was performed at 72° C. for 80 sec. These steps wererepeated 30 cycles to give a 600 bp DNA as a PCR product.

[0133] Addition of europium chloride to the DNA product resulted in thegeneration of extremely strong fluorescence.

EXAMPLE 4 Analysis of liver tissue using labeled PCR product

[0134] A tissue section was prepared from the liver excised from a humaninfected with hepatitis B according to the method described in Example2, except the use of a normal sterilized purified water in the place ofDEPC-treated water, to give an air-dried sample on a slide glass.

[0135] A hybridization solution consisting of 50% formamide, 10 mMTris-HCl (pH 7.6), 10% dextran sulfate, 600 mM NaCl, 0.25% SDS, 1 mMEDTA, 200 μg/ml tRNA and 1×Denhardt's solution,was prepared. Prior tohybridization, the air-dried sample was prehybridized with thishybridization solution without PCR amplification product for 2 hr.

[0136] The labeled PCR amplification product obtained in Example 3 wasdissolved in this hybridization solution to the concentration of 70ng/ml in a DNA content. The mixture was preincubated at 85° C. for 10min, and diluted 10-fold with the hybridization solution. Thishybridization solution (about 40 μl) was added dropwise onto saidair-dried sample on the slide glass. The slide glass was covered with aparafilm and heated at 95° C. for 2 min on a hotplate.

[0137] This slide glass was incubated at 37° C. for 16 hr in a moisturechamber, in which a paper towel moistened with 50% formamide solutionwas set, to allow the air-dried sample to hybridize with the labeled PCRamplification product.

[0138] After hybridization, the parafilm was removed from the slideglass in 5×SSC solution (40° C.), and the slide glass was heated in2×SSC and 50% formamide at 40° C. for 30 min. Then, the slide glass waswashed with TNE solution, and reacted with 5 μg/ml RNase in TNE solutionfor 10 min. The slide glass was washed successively with 2×SSC at 40° C.for 20 min (once), and with 0.2×SSC at 40° C. for 20 min (twice). Theslide glass was immersed in 0.2×SSC solution (pH 8.5) containing 0.1 mMeuropium chloride, and the tissue section on the slide glass wasobserved with a fluorescence microscope.

[0139] As a result, a mosaic staining pattern was detected in livercells in the lobulus on the slide glass. As a comparative example,hybridization was tried using FITC in the place of the labeled compoundmentioned above. However, the fluorescence of FITC was significantlydegraded when an FITC-labeled PCR product was obtained, so that thesubsequent hybridization step could not be performed.

[0140] Thus, as an alternative, a DNA product was obtained byamplification using unlabeled oligonucleotides having the nucleotidesequences depicted in SEQ ID: NO: 1 and SEQ ID: NO: 2 as a pair ofprimers and a nucleic acid fraction derived from an HBsAg stronglypositive patient-derived serum as a template. Said amplification productwas reacted with FITC to give a fluorescence-labeled probe, which wassubjected to hybridization, wherein no europium chloride was added.Hybridization using said FITC-labeled probe gave very weak signals,which showed that the FITC-labeled prove was obviously inferior to thelabeled probe of the present invention.

EXAMPLE 5 Analysis of human pancreas tissue using labeled human insulin

[0141] A labeled human insulin was prepared using a standard humaninsulin (Sigma) in the same manner as in Example 1.

[0142] A pancreas tissue excised from a human was treated in the samemanner as in Example 2 to give a section. This section was placed on aslide glass and immersed in 4% formamide solution at room temperaturefor 10 min. To this section was added TBS solution [Tris-NaCl buffer (pH7.6), 50 μl] containing 5% skim milk. The section was heated at 37° C.for 2 hr, and washed with TBS solution (pH 7.6) 3 times.

[0143] As a hybridization solution, a solution of 1 mM EDTA and 0.2% BSAin TBS solution (pH 7.6) was prepared.

[0144] The labeled human insulin was dissolved in this hybridizationsolution to the concentration of 10 ng/ml, and the mixture (about 40 μl)was dropped onto the pancreas tissue section. The slide glass wascovered with parafilm, and incubated at 37° C. for 8 hr in a moisturechamber in which a paper towel moistened with 50% formamide solution wasset, to allow the section to hybridize with the labeled probe.

[0145] After hybridization, the parafilm was removed from the slideglass in 5×SSC solution (40° C.), and the slide glass was heated in2×SSC and 50% formamide at 40° C. for 30 min. Then, the slide glass waswashed with TNE solution, and reacted with 5 μg/ml RNase in TNE solutionfor 10 min. The slide glass was washed successively with 2×SSC at 40° C.for 20 min (once), and with 0.2×SSC at 40° C. for 20 min (twice). Theslide glass was immersed in 0.2×SSC solution (pH 8.5) containing 0.1 mMeuropium chloride, and the tissue section on the slide glass wasobserved with a fluorescence microscope.

[0146] As a result, signals derived from the fluorescent complex of thelabeled human insulin and europium chloride were detected on the tissuesection.

[0147] When hybridization was performed using an anti-human insulinreceptor antibody (Austral Biologicals (ABI)) labeled with rhodamine inthe place of the labeled human insulin mentioned above as a comparativeexample, almost the same level of fluorescence image was obtained.

[0148] Accordingly, it was concluded that the labeled human insulin ofthe invention specifically reacted with a human insulin receptor.

EXAMPLE 6 Analysis of chromosome preparation derived from peripheralblood cell using denatured DNA probe

[0149] (Culture of Peripheral Lymphocyte)

[0150] Sterilely obtained peripheral blood supplemented with heparin (1ml) and RPMI1640 medium (GIBCO BRL, 9 ml) supplemented with 15% fetalcalf serum were mixed, and transferred into a culture flask.Phytohemagglutinin (Welcome) was added to the final concentration of 10g/ml, and the peripheral blood was cultured in a CO₂ incubator with 5%CO₂ atmosphere at 37° C. After 48 hrs of culture, thymidine (Sigma) wasadded to the final concentration of 300 μg/ml, and the culture wascontinued. At 63 hr after the start of the culture, peripheral bloodcells comprising lymphocytes were transferred to a new tube, andcentrifuged at 1,200 rpm for 5 min. The cells were rinsed by addingRPMI1640 medium (10 ml) to the tube and gently stirring. Said rinsingstep was repeated once. RPMI1640 medium supplemented with 15% fetal calfserum (10 ml) and the peripheral blood cells comprising lymphocytes weremixed, and cultured. At 63.5 hr after the start of the culture,bromodeoxyuridine (Sigma) was added to the final concentration of 50ng/ml, the mixture was stirred, and the culture was continued. At 70 hrafter the start of the culture, the peripheral blood cells comprisinglymphocytes were harvested by centrifugation at 1,200 rpm.

[0151] (Preparation of Chromosome)

[0152] To the harvested peripheral blood cells was added 0.075 M KCl (10ml), and the suspension was stood at room temperature for 30 min. Afterhypotonization, the suspension was centrifuged at 1,200 rpm for 5 min.The supernatant (about 3 ml) and the precipitate were sufficientlystirred using a Pasteur pipette, and gradually dropped into methanol:acetic acid (3:1, carnoy solution, 10 ml). The mixture was sufficientlystirred, stood for 10 min and centrifuged at 1,200 rpm for 5 min. Thesupernatant was removed. To the precipitate was added a fresh carnoysolution (10 ml), and the both were mixed with stirring. This step ofwashing with carnoy solution was repeated twice. The concentration ofthe suspension was adjusted with caroy solution. The suspension wasdropped onto the center of a slide glass, which was followed by steamfixation using a pot containing boiled water. The fixed sample on theslide glass was dried at 37° C. overnight, which was followed byadhesion in a dry heat sterilizer at 65° C. for 4 hr to give achromosome preparation. This chromosome preparation was stained with2×SSC containing 1 μg/ml fluorescent dye, Hoechst 33258 (Molecularprobe) for 5 min, gently rinsed with 2×SSC and covered with a coverglass, which was followed by standing on a hot plate (75° C.) for 3 min.The preparation was exposed to UV light on the hot plate at a distanceof 1 cm from the preparation with a black light (20 W, Toshiba). Thecover glass on the slide glass was removed and the slide glass wasrinsed twice with distilled water, dried and stored at −20° C. with thechromosome preparation carried thereon.

[0153] (Preparation of Labeled dUTP)

[0154] A labeled dUTP was prepared using dUTP (deoxy UTP, Toyo Boseki)in the same manner as in Example 1.

[0155] This labeled dUTP and KRAS ONCOGENE (Lab Logics, large probe)were subjected to nick translation method to give a labeled nucleicacid.

[0156] The nick translation followed the protocol using a nicktranslation kit manufactured by Boehringer. The labeled dUTP was used ata final concentration of 0.05 mM.

[0157] After the nick translation, 4 M ammonium acetate (2.5 μl), 10mg/ml salmon sperm DNA (Sigma, 2.0 μl), 10 mg/ml E. coli tRNA (Sigma,2.0 μl) and special grade ethanol (75 μl) were added to the nicktranslation reaction mixture (20 μl), admixed well, stored at −80° C.for 1 hr, and centrifuged at 15,000 rpm to give precipitate, which wasstirred in special grade formamide to dissolution.

[0158] (Hybridization)

[0159] To the labeled nucleic acid (5 μl) prepared according to theabove-mentioned method was added 10 mg/ml Cot-1 DNA (manufactured byVysis, 5 μl) and incubated in a heat block at 70° C. for 10 min todenature the labeled probe to give a denatured DNA probe, which wasquickly cooled in ice water.

[0160] Then, the above-mentioned slide glass carrying the chromosomepreparation was immersed in a coupling jar filled with 70% formamide(2×SSC) at 70° C. to denature the sample with heat. The sample wasimmediately moved into 70% ethanol at −20° C., rapidly cooled for 2 min,dehydrated with 100% ethanol and dried. The above-mentioned denaturedDNA probe was mixed in a solution having a final concentration of 50%formamide and 10% dextran sulfate (in 2×SSC) and placed on theabove-mentioned chromosome preparation. The denatured DNA probe solutionwas uniformly spread thereon using a parafilm strip to prevent inclusionof air foams. The chromosome sample on the slide glass and the denaturedDNA probe were hybridized in a sealed moistened chamber containing afilter paper impregnated with 2×SSC on the bottom therein at 37° C. for18 hr.

[0161] The parafilm was stripped off the slide glass and the slide glasswas immersed in a coupling jar filled with 50% formamide (in 2×SSC) at37° C. and rinsed for 15 min. This slide glass was stood still in 2×SSC(room temperature) for 1 min and then stood still for 15 min in 1×SSC(room temperature) and 5 min in 4×SSC (room temperature). Then, 2×SSC(pH 8.5) containing 0.1 mM europium chloride was dropped on a slideglass and the tissue section strip on the slide glass was observed witha fluorescence microscope.

[0162] As a result, the 11th chromosome was found to have fluorescence,which coincided with the localization of nucleic acid containing KRASONCOGENE, thereby confirming possible specific detection.

EXAMPLE 7

[0163] (Preparation of Fluorescence-labeled Streptoavidin)

[0164] Recombinant streptoavidin (24 mg, Boehringer Mannheim) wasdissolved in 100 mM carbonate buffer (pH 9.3, 2 ml) and dialyzed againstthe same buffer. From the absorbance of dialysis solution at 280 nm, itwas confirmed to have a 3.4 mg/ml protein concentration. To the entireamount of 2 ml thereof was dropwise added an anhydrous DMF solution (0.4ml) containing the labeled compound (7.4 mg) of Reference example 3 andthe mixture was stirred at 25° C. for 1 hr. After stirring, the reactionmixture was eluted with 50 mM ammonium carbonate using Sephadex G-50column (about 30 ml bed) to separate a non-reacted labeled compound.From the absorption coefficient 3.41×10⁴ (cm⁻¹M⁻¹) at 330 nm of theprotein fraction and the molecular weight of recombinant streptoavidinof about 52,000, the cross-linked labeled compound was calculated to beabout 20 molecules per 1 molecule of streptoavidin. Sodium azide wasadded to the protein fraction to the concentration of 0.1%, adjusted topH 6.5 with 1N HCl and stored at 4° C.

[0165] (Preparation of DNA Probe Using Nick Translation Kit)

[0166] Using the KRAS ONCOGENE (Lab Logics, large probe) used in Example6 and biotin-21-dUTP nick translation kit (Clontech), and following theprotocol attached to the kit, biotin-21-dUTP was incorporated into thenucleic acid KRAS ONCOGENE to give a biotinylated KRAS ONCOGENE DNAprobe. The biotin-21-dUTP has the following structure including alinkage group.

[0167] wherein R has the formula

[0168] To the reaction mixture (20 μl) after nick translation reactionwere added 4 M ammonium acetate (2.5 μl), 10 mg/ml salmon sperm DNA(Sigma, 2.0 μl), 10 mg/ml E. coli tRNA (Sigama, 2.0 μl) and specialgrade ethanol (75 μl) and admixed well. After storing at −80° C. for 1hr, it was centrifuged at 15,000 rpm and the precipitate was thoroughlystirred in special grade formamide to dissolution.

[0169] (Hybridization)

[0170] Completely in the same manner as in Example 6, a denatured DNAprobe was prepared and hybridized with a chromosome preparation.

[0171] After hybridization, a parafilm was stripped off the slide glassand the slide glass was immersed in a coupling jar filled with 50%formamide (in 2×SSC) at 37° C. and rinsed for 15 min. This slide glasswas stood still in 2×SSC (room temperature) for 1 min and then stoodstill for 15 min in 1×SSC (room temperature) and 5 min in 4×SSC (roomtemperature). The labeled streptoavidin prepared above was diluted with2×SSC to the concentration of 0.02 mg/ml. The slide glass was leftstanding still in the solution at room temperature for 15 min. Using1×SSC (room temperature), the slide glass was left standing still for 5min, which step was repeated three times. Then, 2×SSC (pH 8.5)containing 0.1 mM europium chloride was dropped on a slide glass and thetissue section strip on the slide glass was observed with a fluorescencemicroscope.

[0172] As a result, the 11th chromosome was found to have fluorescencelike Example 6, but apparently had stronger fluorescence. Thisfluorescence intensity was considered to reflect the high incorporationrate of biotinylated-21-UTP into the nucleic acid and the great numberof the labeled compounds bonded to streptoavidin. The synchronizedlocalization with KRAS ONCOGENE was confirmed and the method wasconcluded to be a specific detection method.

[0173] This application is based on patent application Nos. 119768/1998and 184852/1998 filed in Japan, the contents of which are herebyincorporated by reference.

1 2 1 27 DNA Artificial Sequence Oligonucleotide designed to act assense primer for amplifying HBsAg gene. 1 catggagaac atcacacatc aggattc27 2 24 DNA Artificial Sequence Oligonucleotide designed to act asantisense primer for amplifying HBsAg gene. 2 aatgtatacc cagagacaaa acaa24

What is claimed is:
 1. A method for analyzing an objective substance,comprising reacting a labeled probe with an objective substance on abiological sample, said probe comprising a label substance of theformula (I):

wherein A¹ is an aromatic group, R¹is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II):

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, bonded to a probeselected from the group consisting of nucleic acid, nucleic acid bindingprotein, low molecular ligand and receptor for ligand (except antibody)via a cross-linking group or a cross-linking group and a conjugatinggroup, adding a heavy metal ion and assaying fluorescence of theresultant fluorescent complex.
 2. A method for analyzing an objectivesubstance, comprising adding a heavy metal ion to a labeled probe, saidprobe comprising a label substance of the formula (I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II):

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, bonded to a probeselected from the group consisting of nucleic acid, nucleic acid bindingprotein, low molecular ligand and receptor for ligand (except antibody)to give a fluorescent complex, reacting the complex with an objectivesubstance on a biological sample and assaying fluorescence of theresultant fluorescent complex.
 3. The analysis method of claim 1 orclaim 2, wherein the objective substance is a member selected from thegroup consisting of a nucleic acid, a nucleic acid-bound protein, a lowmolecular weight ligand and a ligand receptor.
 4. The analysis method ofany of claim 1 to claim 3, wherein the biological sample is a memberselected from the group consisting of a tissue, a cell or a chromosome.5. The analysis method of claim 1 or claim 2, wherein the cross-linkinggroup is a sulfonyl group or a carbonyl group.
 6. The analysis method ofclaim 1 or claim 2, wherein the conjugating group binds a cross-linkinggroup and a probe.
 7. The analysis method of claim 1 or claim 2, whereinthe conjugating group is a divalent aliphatic hydrocarbon having 5 to 25carbon atoms and optionally having 7 or less amide bonds betweencarbons.
 8. The analysis method of claim 1 or claim 2, wherein theconjugating group comprises an affinity-bound biotin and avidin.
 9. Theanalysis method of claim 8, wherein the biotin is amide-bound with adivalent aliphatic hydrocarbon having 5 to 25 carbon atoms andoptionally having 7 or less amide bonds between carbons.
 10. Theanalysis method of claim 9, wherein the aliphatic hydrocarbon is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂CH₂—NH)²⁻ which is bonded toa probe.
 11. A labeled nucleic acid probe comprising a label substanceof the formula (I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II):

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, bonded to a nucleicacid probe via a cross-linking group.
 12. The labeled nucleic acid probeof claim 11, wherein the label substance has the formula (III):

wherein n is an integer of 1-6.
 13. The labeled nucleic acid probe ofclaim 11 or claim 12, wherein the cross-linking group is a sulfonylgroup or a carbonyl group.
 14. The labeled nucleic acid probe of any ofclaim 11 to claim 13, wherein the label substance is bonded to nucleicacid probe via a conjugating group.
 15. The labeled nucleic acid probeof claim 14, wherein the conjugating group is a divalent aliphatichydrocarbon having 5 to 25 carbon atoms and optionally having 7 or lessamide bonds between carbons.
 16. The labeled nucleic acid probe of claim15, wherein the conjugating group has the formula (IV):

wherein a is an integer of 0-6 and b is 0 or
 1. 17. The labeled nucleicacid probe of claim 14, wherein the conjugating group comprises anaffinity-bound biotin and an avidin.
 18. The labeled nucleic acid probeof claim 17, wherein the biotin is amide-bound with a divalent aliphatichydrocarbon having 5 to 25 carbon atoms and optionally having 7 or lessamide bonds between carbons.
 19. The labeled nucleic acid probe of claim18, wherein the aliphatic hydrocarbon is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂- which is bonded toa probe.
 20. A fluorescent complex comprising the labeled nucleic acidprobe of any of claim 11 to claim 19 and a heavy metal ion.
 21. Thefluorescent complex of claim 20, wherein the heavy metal ion is alanthanoide metal ion or radium ion.
 22. The fluorescent complex ofclaim 21, wherein the lanthanoide metal ion is a member selected fromthe group consisting of ions of europium, samarium, terbium, dysprosiumand a mixture thereof.
 23. A reagent for analyzing a nucleic acid,comprising the labeled nucleic acid probe of any of claim 11 to claim19.
 24. The reagent for analyzing of claim 23, comprising a heavy metalion.
 25. The reagent for analyzing of claim 24, wherein the heavy metalion is a lanthanoide metal ion or radium ion.
 26. A labeled nucleotidecomprising a label substance of the formula (I):

wherein A¹ is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, which is bonded to anucleotide via a cross-linking group.
 27. The labeled nucleotide ofclaim 26, wherein the nucleotide is dUTP.
 28. The labeled nucleotide ofclaim 26 or claim 27, wherein the cross-linking group is a sulfonylgroup orcarbonyl group.
 29. The labeled nucleotide of any of claim 26 toclaim 28, wherein the label substance is bonded to a nucleotide via aconjugating group.
 30. The labeled nucleotide of claim 29, wherein theconjugating group is a divalent aliphatic hydrocarbon having 5 to 25carbon atoms and optionally having 7 or less amide bonds betweencarbons.
 31. The labeled nucleotide of claim 30, wherein the conjugatinggroup has the formula (IV):

wherein a is an integer of 0-6 and b is 0 or
 1. 32. The labelednucleotide of claim 29, wherein the conjugating group comprises anaffinity-bound biotin and avidin.
 33. The labeled nucleotide of claim32, wherein the biotin is amide-bound with a divalent aliphatichydrocarbon having 5 to 25 carbon atoms and optionally having 7 or lessamide bonds between carbons.
 34. The labeled nucleotide of claim 32,wherein the aliphatic hydrocarbon is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)₂ which is bonded toa probe.
 35. A fluorescent complex comprising the labeled nucleotide ofany of claim 26 to claim 34 and a heavy metal ion.
 36. The fluorescentcomplex of claim 35, wherein the heavy metal ion is a lanthanoide metalion or radium ion.
 37. The fluorescent complex of claim 36, wherein thelanthanoide metal ion is a member selected from the group consisting ofions of europium, samarium, terbium, dysprosium and a mixture thereof.38. A method for producing a labeled nucleic acid probe comprisingreacting the labeled nucleotide of any of claim 26 to claim 34, dNTPsand a single strand DNA in the presence of a primer and a DNApolymerase.
 39. A method for producing a labeled nucleic acid probecomprising reacting the labeled nucleotide of any of claim 26 to claim34, dNTPs and a double stranded DNA in the presence of 5′-exonuclease,DNase and a DNA polymerase.
 40. A labeled nucleic acid probe obtained bythe production method of claim 38) or claim
 39. 41. A reagent foranalyzing nucleic acid comprising a label substance of the formula (I):

wherein A¹ is an aromatic group, R¹is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II):

wherein A² and A³are the same or different and each is an aromaticgroup, R²and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, to which avidin iscovalently bonded via a cross-linking group, and a nucleotide to whichbiotin is bonded via a linkage group.
 42. The reagent for analysis ofnucleic acid of claim 41, comprising a dNTP primer, a DNA polymerase anda heavy metal.
 43. The reagent for analysis of nucleic acid of claim 41,comprising dNTPs, 5′-exonuclease, DNase, a DNA polymerase and a heavymetal.
 44. The reagent for analysis of nucleic acid of claim 41, whereinthe cross-linking group is a sulfonyl group or carbonyl group.
 45. Thereagent for analysis of nucleic acid of claim 41, wherein the nucleotideis dUTP.
 46. A method for analyzing an objective substance comprisingreacting the objective substance with a nucleic acid probe comprising anucleotide to which biotin is bonded via a linkage group as a componenton a biological sample and then with a the label substance of theformula (I):

wherein A¹is an aromatic group, R¹ is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, or a label substanceof the following formula (II):

wherein A² and A³ are the same or different and each is an aromaticgroup, R² and R³ are the same or different and each is a hydrogen or—COCH₂COC_(n)F_(2n+1) and n is an integer of 1-6, to which avidin iscovalently bonded via a crosslinking group, adding a heavy metal ion andassaying fluorescence of the resultant fluorescent complex.
 47. Themethod for analysis of claim 46, wherein the nucleic acid probe isobtained by reacting a nucleotide to which biotin is bonded via alinkage group, dNTPs and a single strand DNA in the presence of a primerand a DNA polymerase.
 48. The method for analysis of claim 46, whereinthe nucleic acid probe is obtained by reacting a nucleotide to whichbiotin is bonded via a linkage group, dNTPs and a double stranded DNA inthe presence of 5′-exonuclease, DNase and a DNA polymerase.
 49. Themethod for analysis of claim 46, wherein the linkage group is a divalentaliphatic hydrocarbon having 5 to 25 carbon atoms and optionally having7 or less amide bonds between carbons.
 50. The method for analysis ofclaim 49, wherein the aliphatic hydrocarbon is—CH═CH—CO—NH—CH₂—CH₂—NH—(CO—CH₂—CH₂—CH₂—CH₂—CH₂—NH)_(2—).
 51. The methodfor analysis of claim 46, wherein the cross-linking group is a sulfonylgroup or carbonyl group.
 52. The method for analysis of claim 46,wherein the nucleotide is dUTP.